ChIP sonication problem - (Dec/27/2007 )

Hi,

I was trying to optimize DNA shearing for CHIP assay but got some problem with sonication.

I used Branson Sonifier 250 for shearing DNA. The DNA samples were extracted according to the UPState CHIP protocol. For sonication, I used different settings: (1) 30% duty cycle, output setting 3, 5 or 10 second pulse, 5 pulses, 60 second between each pulse; (2) 40% duty cycle, output setting 4, 5 or 10 second pulse, 3 or 5 pulses, 60 second between each pulse; (3) 100% duty cycle, output setting 2, 5 or 10 second pulse, 3 or 5 pulses, 60 second between each pulse. For all these conditions, the bands I saw on the agarose gel were located around 100bp. There were two bands, one near 100bp and the other one near 150bp. I don't know why the different sonication conditions all generated DNAs to 100bps. Has anyone encountered similar problems before? What might cause this to happen?

I didn't have any CHIP experience before, any suggestion would be appreciated.

Thank you first!

esmile

-esmile-

Are you sure that what you are seeing at 100bp is actually DNA? Did you treat the DNA with RNase prior to running on the gel?

-KPDE-

QUOTE

Are you sure that what you are seeing at 100bp is actually DNA? Did you treat the DNA with RNase prior to running on the gel?

No, I didn't treat the samples with RNase. Are you suggesting that the 100bp bands might be RNA?Thanks

-esmile-

Hello esmile,

I agree the small band may be RNA. You have to reverse crosslink and RNase treat your DNA before running the agarose gel.

I believe the reason you did not get a smear between 0.2-1kb is that the sonication is not enough. I have not used the type of sonicator you are using. With another sonicator and a power setting of 2 on it, for one of my cell I have to do 14 pulses of 10 second sonication. You can keep the output fixed such as at 2 (higher than 2 is prone to cause foaming), and vary the pulse number.

-pcrman-

QUOTE (pcrman @ Dec 27 2007, 08:30 PM)

Hello esmile,

I agree the small band may be RNA. You have to reverse crosslink and RNase treat your DNA before running the agarose gel.

I believe the reason you did not get a smear between 0.2-1kb is that the sonication is not enough. I have not used the type of sonicator you are using. With another sonicator and a power setting of 2 on it, for one of my cell I have to do 14 pulses of 10 second sonication. You can keep the output fixed such as at 2 (higher than 2 is prone to cause foaming), and vary the pulse number.

Thank you pcrman. I will try to RNase treat the samples and see whether it will make a difference. BTW, will a bath sonicator be ok for shearing DNA?

-esmile-

QUOTE (esmile @ Dec 28 2007, 08:02 AM)

QUOTE (pcrman @ Dec 27 2007, 08:30 PM)

Hello esmile,

I agree the small band may be RNA. You have to reverse crosslink and RNase treat your DNA before running the agarose gel.

I believe the reason you did not get a smear between 0.2-1kb is that the sonication is not enough. I have not used the type of sonicator you are using. With another sonicator and a power setting of 2 on it, for one of my cell I have to do 14 pulses of 10 second sonication. You can keep the output fixed such as at 2 (higher than 2 is prone to cause foaming), and vary the pulse number.

Thank you pcrman. I will try to RNase treat the samples and see whether it will make a difference. BTW, will a bath sonicator be ok for shearing DNA?

If, by bath sonicator, you mean an ultrasonic bath, like what is used for critical cleaning of small parts (kind of like an upscale jewelry cleaner) then no. If you are referring to a sonicator with a cup horn (like a Bioruptor) then yes, many people have had success with those.

-KPDE-

QUOTE (KPDE @ Dec 28 2007, 05:42 PM)

QUOTE (esmile @ Dec 28 2007, 08:02 AM)

QUOTE (pcrman @ Dec 27 2007, 08:30 PM)

Hello esmile,

I agree the small band may be RNA. You have to reverse crosslink and RNase treat your DNA before running the agarose gel.

I believe the reason you did not get a smear between 0.2-1kb is that the sonication is not enough. I have not used the type of sonicator you are using. With another sonicator and a power setting of 2 on it, for one of my cell I have to do 14 pulses of 10 second sonication. You can keep the output fixed such as at 2 (higher than 2 is prone to cause foaming), and vary the pulse number.

Thank you pcrman. I will try to RNase treat the samples and see whether it will make a difference. BTW, will a bath sonicator be ok for shearing DNA?

If, by bath sonicator, you mean an ultrasonic bath, like what is used for critical cleaning of small parts (kind of like an upscale jewelry cleaner) then no. If you are referring to a sonicator with a cup horn (like a Bioruptor) then yes, many people have had success with those.

Thank you KPDE. The bath sonicator we have is ultrasonic. Well, guess I have to stick to the tip sonicator and optimize the sonication conditions and add RNase.

-esmile-

I should sell this protocol for cash since so many people have the same problems....but, when testing for shear length; #1 go through the sonicating procedure to decrosslinking, then proteinase K treat.#2 Phenol chloroform extract #3 RNAse treat. #4 EtOH precipitation. This works well. Also, there is a 100% chance that the small bands you see are RNA. Anything you see less than 300-400 basepairs is RNA guaranteed.

PS. To avoid the next 59,000 problems you will have with ChIP, do a positive and negative control I'm not even being sarcastic here, most of the problems people have on this site seem to stem from a lack of understanding of what controls are useful for.